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Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen*

  • Joyce Sayegh
    Affiliations
    Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520
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  • Jian Cao
    Affiliations
    Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520
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  • Mike Ran Zou
    Affiliations
    Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520
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  • Alfonso Morales
    Affiliations
    Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520
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  • Lauren P. Blair
    Affiliations
    Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520
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  • Michael Norcia
    Affiliations
    Yale Center for Molecular Discovery, Yale University, West Haven, Connecticut 06516
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  • Denton Hoyer
    Affiliations
    Yale Center for Molecular Discovery, Yale University, West Haven, Connecticut 06516
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  • Alan J. Tackett
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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  • Jane S. Merkel
    Affiliations
    Yale Center for Molecular Discovery, Yale University, West Haven, Connecticut 06516
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  • Qin Yan
    Correspondence
    To whom correspondence should be addressed: Dept. of Pathology, Yale School of Medicine, 310 Cedar St., BML 348C, P. O. Box 208023, New Haven, CT. Tel.: 203-785-6672; Fax: 203-785-2443;
    Affiliations
    Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520
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  • Author Footnotes
    * This work was supported in part by a pilot grant from the Yale Center for Molecular Discovery, Clinical and Translational Science Award (CTSA) Scholar Award from Yale Center for Clinical Investigation, the Alexander and Margaret Stewart Trust Fellowship, Breast Cancer Alliance Young Investigator Grant, Melanoma Research Foundation Career Development Award, a Developmental Research Award from Yale Specialized Programs of Research Excellence (YSPORE) in Skin Cancer (all to Q. Y.) and by National Institutes of Health Grants CA16359 (to the Yale Comprehensive Cancer Center) and R01 DA025755 (to A. J. T.). This work was made possible by CTSA Grant UL1 RR024139 from the National Center for Advancing Translational Sciences.
    This article contains supplemental Figs. 1–3 and Tables 1–4.
Open AccessPublished:February 13, 2013DOI:https://doi.org/10.1074/jbc.M112.419861
      JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC50 of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.

      Introduction

      Covalent posttranslational modification of histones on lysine tails is essential for gene regulation and DNA repair (
      • Blair L.P.
      • Yan Q.
      Epigenetic mechanisms in commonly occurring cancers.
      ). Histone lysine methylations are now widely accepted modifications for activating or silencing gene transcription, depending on the site and degree of methylation (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ). For example, trimethylated lysine 4 in histone H3 (H3K4me3) is associated with active transcription, whereas trimethylated lysine 27 in histone H3 (H3K27me3) is associated with gene silencing.
      The enzymes responsible for the demethylation of H3K4me3 are the Jumonji AT-rich interactive domain 1 (JARID1)
      The abbreviations used are: JARID1
      Jumonji AT-rich interactive domain 1
      KDM5
      lysine demethylase 5
      JmjC
      Jumonji
      2,4-PDCA
      2,4-pyridinedicarboxylic acid monohydrate
      α-KG
      α-ketoglutarate
      bio-
      biotinylated
      DMSO
      dimethyl sulfoxide
      HER2+
      HER2-positive
      PBIT
      2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one
      UTX
      ubiquitously transcribed tetratricopeptide repeat protein X-linked
      JMJD3
      JmjC domain-containing protein 3.
      or lysine demethylase 5 (KDM5) family of lysine demethylases (
      • Klose R.J.
      • Yan Q.
      • Tothova Z.
      • Yamane K.
      • Erdjument-Bromage H.
      • Tempst P.
      • Gilliland D.G.
      • Zhang Y.
      • Kaelin Jr., W.G.
      The retinoblastoma binding protein RBP2 is an H3K4 demethylase.
      ,
      • Lee M.G.
      • Norman J.
      • Shilatifard A.
      • Shiekhattar R.
      Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein.
      ,
      • Christensen J.
      • Agger K.
      • Cloos P.A.
      • Pasini D.
      • Rose S.
      • Sennels L.
      • Rappsilber J.
      • Hansen K.H.
      • Salcini A.E.
      • Helin K.
      RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3.
      ,
      • Iwase S.
      • Lan F.
      • Bayliss P.
      • de la Torre-Ubieta L.
      • Huarte M.
      • Qi H.H.
      • Whetstine J.R.
      • Bonni A.
      • Roberts T.M.
      • Shi Y.
      The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases.
      ,
      • Secombe J.
      • Li L.
      • Carlos L.
      • Eisenman R.N.
      The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth.
      ,
      • Yamane K.
      • Tateishi K.
      • Klose R.J.
      • Fang J.
      • Fabrizio L.A.
      • Erdjument-Bromage H.
      • Taylor-Papadimitriou J.
      • Tempst P.
      • Zhang Y.
      PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation.
      ). This family consists of JARID1A (also known as KDM5A or RBP2), JARID1B (also known as KDM5B or PLU1), JARID1C (also known as KDM5C or SMCX), and JARID1D (also known as KDM5D or SMCY) in mammals (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ). Similar to other Jumonji C (JmjC) domain-containing demethylases, the JARID1 enzymes catalyze the demethylation of histones in an iron(II) and α-ketoglutarate (α-KG)-dependent reaction (
      • Klose R.J.
      • Zhang Y.
      Regulation of histone methylation by demethylimination and demethylation.
      ). In this reaction, the oxidative decarboxylation of α-KG results in a hydroxylated methyl-lysine intermediate, which is thermodynamically unstable. The release of the hydroxyl and methyl groups as formaldehyde from this intermediate results in a demethylated lysine residue. Although all the JmjC domain histone demethylases catalyze the reaction via a similar mechanism, they clearly demonstrate specificity toward particular lysine residue(s) (
      • Hou H.
      • Yu H.
      Structural insights into histone lysine demethylation.
      ).
      The JARID1 demethylases have been linked to human diseases such as cancer and X-linked mental retardation (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ). Both JARID1A and JARID1B are potential oncoproteins, and both are overexpressed in a variety of cancers (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ). Increased expression of JARID1A promotes a more stem-like phenotype and enhanced resistance to anticancer agents (
      • Sharma S.V.
      • Lee D.Y.
      • Li B.
      • Quinlan M.P.
      • Takahashi F.
      • Maheswaran S.
      • McDermott U.
      • Azizian N.
      • Zou L.
      • Fischbach M.A.
      • Wong K.K.
      • Brandstetter K.
      • Wittner B.
      • Ramaswamy S.
      • Classon M.
      • Settleman J.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ). Moreover, loss of JARID1A inhibits tumorigenesis in two genetically engineered mouse cancer models (
      • Lin W.
      • Cao J.
      • Liu J.
      • Beshiri M.L.
      • Fujiwara Y.
      • Francis J.
      • Cherniack A.D.
      • Geisen C.
      • Blair L.P.
      • Zou M.R.
      • Shen X.
      • Kawamori D.
      • Liu Z.
      • Grisanzio C.
      • Watanabe H.
      • Minamishima Y.A.
      • Zhang Q.
      • Kulkarni R.N.
      • Signoretti S.
      • Rodig S.J.
      • Bronson R.T.
      • Orkin S.H.
      • Tuck D.P.
      • Benevolenskaya E.V.
      • Meyerson M.
      • Kaelin Jr., W.G.
      • Yan Q.
      Loss of the retinoblastoma binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rb1 or Men1.
      ). JARID1B is highly expressed in human mammary tumors and breast cancer cell lines, but not in normal adult breast tissue (
      • Lu P.J.
      • Sundquist K.
      • Baeckstrom D.
      • Poulsom R.
      • Hanby A.
      • Meier-Ewert S.
      • Jones T.
      • Mitchell M.
      • Pitha-Rowe P.
      • Freemont P.
      • Taylor-Papadimitriou J.
      A novel gene (PLU-1) containing highly conserved putative DNA/chromatin binding motifs is specifically up-regulated in breast cancer.
      ). Knockdown of JARID1B leads to up-regulation of tumor suppressor genes including BRCA1 (
      • Yamane K.
      • Tateishi K.
      • Klose R.J.
      • Fang J.
      • Fabrizio L.A.
      • Erdjument-Bromage H.
      • Taylor-Papadimitriou J.
      • Tempst P.
      • Zhang Y.
      PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation.
      ). Down-regulation of JARID1B in breast cancer cells decreased tumor formation potential of these cells in mouse syngeneic or xenograft models (
      • Yamane K.
      • Tateishi K.
      • Klose R.J.
      • Fang J.
      • Fabrizio L.A.
      • Erdjument-Bromage H.
      • Taylor-Papadimitriou J.
      • Tempst P.
      • Zhang Y.
      PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation.
      ,
      • Catchpole S.
      • Spencer-Dene B.
      • Hall D.
      • Santangelo S.
      • Rosewell I.
      • Guenatri M.
      • Beatson R.
      • Scibetta A.G.
      • Burchell J.M.
      • Taylor-Papadimitriou J.
      PLU-1/JARID1B/KDM5B is required for embryonic survival and contributes to cell proliferation in the mammary gland and in ER+ breast cancer cells.
      ). JARID1B is also up-regulated in advanced and metastatic prostate tumors (
      • Xiang Y.
      • Zhu Z.
      • Han G.
      • Ye X.
      • Xu B.
      • Peng Z.
      • Ma Y.
      • Yu Y.
      • Lin H.
      • Chen A.P.
      • Chen C.D.
      JARID1B is a histone H3 lysine 4 demethylase up-regulated in prostate cancer.
      ) and is required for continuous growth of melanoma cells (
      • Roesch A.
      • Fukunaga-Kalabis M.
      • Schmidt E.C.
      • Zabierowski S.E.
      • Brafford P.A.
      • Vultur A.
      • Basu D.
      • Gimotty P.
      • Vogt T.
      • Herlyn M.
      A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.
      ). Taken together, both JARID1A and JARID1B enzymes are very attractive targets for cancer therapy (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ). Even so, no specific inhibitor of these two epigenetic regulators is currently available, and the development of small molecule inhibitors is in demand.
      Until now, no high throughput screen has been reported for the JARID1 family of histone lysine demethylases. Small molecule inhibitor screens of other JmjC domain-containing demethylases employed methods including detection of the reaction byproduct formaldehyde (
      • Sakurai M.
      • Rose N.R.
      • Schultz L.
      • Quinn A.M.
      • Jadhav A.
      • Ng S.S.
      • Oppermann U.
      • Schofield C.J.
      • Simeonov A.
      A miniaturized screen for inhibitors of Jumonji histone demethylases.
      ,
      • King O.N.
      • Li X.S.
      • Sakurai M.
      • Kawamura A.
      • Rose N.R.
      • Ng S.S.
      • Quinn A.M.
      • Rai G.
      • Mott B.T.
      • Beswick P.
      • Klose R.J.
      • Oppermann U.
      • Jadhav A.
      • Heightman T.D.
      • Maloney D.J.
      • Schofield C.J.
      • Simeonov A.
      Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.
      ), mass spectrometry (
      • Rose N.R.
      • Woon E.C.
      • Kingham G.L.
      • King O.N.
      • Mecinović J.
      • Clifton I.J.
      • Ng S.S.
      • Talib-Hardy J.
      • Oppermann U.
      • McDonough M.A.
      • Schofield C.J.
      Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches.
      ), AlphaScreen (
      • Kawamura A.
      • Tumber A.
      • Rose N.R.
      • King O.N.
      • Daniel M.
      • Oppermann U.
      • Heightman T.D.
      • Schofield C.
      Development of homogeneous luminescence assays for histone demethylase catalysis and binding.
      ), and LANCE Ultra and AlphaLISA assays (
      • Gauthier N.
      • Caron M.
      • Pedro L.
      • Arcand M.
      • Blouin J.
      • Labonté A.
      • Normand C.
      • Paquet V.
      • Rodenbrock A.
      • Roy M.
      • Rouleau N.
      • Beaudet L.
      • Padrós J.
      • Rodriguez-Suarez R.
      Development of homogeneous nonradioactive methyltransferase and demethylase assays targeting histone H3 lysine 4.
      ). In these studies, α-KG analogues were reported to inhibit the JmjC demethylases (
      • Suzuki T.
      • Miyata N.
      Lysine demethylases inhibitors.
      ). One such analogue, 2,4-pyridinedicarboxylic acid (2,4-PDCA), has been shown to inhibit the catalytic core of JARID1B (
      • Kristensen L.H.
      • Nielsen A.L.
      • Helgstrand C.
      • Lees M.
      • Cloos P.
      • Kastrup J.S.
      • Helin K.
      • Olsen L.
      • Gajhede M.
      Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.
      ). However, the specificity is likely compromised as these analogues may inhibit other Fe(II)- and α-KG-dependent enzymes, such as prolyl hydroxylases (
      • Suzuki T.
      • Miyata N.
      Lysine demethylases inhibitors.
      ).
      Here we describe a high throughput screen to identify small molecule inhibitors of full-length JARID1B using the AlphaScreen platform. By implementing AlphaScreen technology, we developed a very sensitive assay for detecting demethylation of a biotinylated (bio-) H3K4me3 peptide in vitro. We screened JARID1B against a diverse library consisting of 15,134 molecules and identified several compounds that yielded low μm IC50 values. One novel inhibitor, PBIT, inhibits JARID1B up to 95%, with an IC50 value of about 3 μm. This compound can also inhibit other members of the JARID1 family. It, however, did not inhibit the H3K27me3 demethylases ubiquitously transcribed tetratricopeptide repeat protein, X-linked (UTX) or JmjC domain-containing protein 3 (JMJD3), indicating that PBIT is specific for the JARID1 enzymes. Furthermore, this small molecule is able to modulate H3K4me3 levels in cells and attenuate proliferation of UACC-812 breast cancer cells. Taken together, these studies reveal the identification of novel inhibitors of JARID1B in vitro with therapeutic implications for breast cancer.

      DISCUSSION

      The current study is the first report of a high throughput screen for inhibitors of the JARID1 family of demethylases. In this study, we first characterized full-length JARID1B. We showed that the apparent Km for bio-H3K4me3 is 15 nm. This is much lower than the reported Km for the JARID1B catalytic core (
      • Kristensen L.H.
      • Nielsen A.L.
      • Helgstrand C.
      • Lees M.
      • Cloos P.
      • Kastrup J.S.
      • Helin K.
      • Olsen L.
      • Gajhede M.
      Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.
      ), suggesting that other domains of JARID1B contribute to folding of the protein or substrate recognition and can be targeted for inhibition. We developed a very robust high throughput screen using the AlphaScreen platform to search for novel small molecule inhibitors of the histone lysine demethylase JARID1B. The signal-to-noise ratio was high (∼17), even with only 4 nm enzyme, producing a Z′ factor of ∼0.8 (supplemental Table 4). This allows for usage of small amounts of enzymes and for the identification of inhibitors with very low IC50 values. After screening over 15,000 small molecules, we identified over 90 validated compounds that inhibit JARID1B activity (supplemental Table 2), many of which have IC50 values in the low micromolar range.
      Several types of JmjC demethylase inhibitors have been identified previously, including α-KG analogues, methyl-lysine analogues, 2,4-PDCA, 8-hydroxyquinoline, catechols, Ni(II), bipyridine, NCDM-32, disulfiram analogues, and hydroxamic acids (
      • Suzuki T.
      • Miyata N.
      Lysine demethylases inhibitors.
      ). In this screen, we also identified many of these known JmjC demethylase inhibitors. For example, several of the hits here were identified in the miniaturized screen for inhibitors of the H3K9 demethylase JMJD2E with similar IC50 values (Table 1) (
      • Sakurai M.
      • Rose N.R.
      • Schultz L.
      • Quinn A.M.
      • Jadhav A.
      • Ng S.S.
      • Oppermann U.
      • Schofield C.J.
      • Simeonov A.
      A miniaturized screen for inhibitors of Jumonji histone demethylases.
      ), suggesting that these are nonspecific demethylase inhibitors. Some of these structures contain catechols, which are likely iron chelators and thus nonspecific inhibitors (
      • Baell J.B.
      • Holloway G.A.
      New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays.
      ). Another potent hit, 2,4-PDCA, was identified as an inhibitor for multiple demethylases (
      • King O.N.
      • Li X.S.
      • Sakurai M.
      • Kawamura A.
      • Rose N.R.
      • Ng S.S.
      • Quinn A.M.
      • Rai G.
      • Mott B.T.
      • Beswick P.
      • Klose R.J.
      • Oppermann U.
      • Jadhav A.
      • Heightman T.D.
      • Maloney D.J.
      • Schofield C.J.
      • Simeonov A.
      Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.
      ,
      • Rose N.R.
      • Ng S.S.
      • Mecinović J.
      • Liénard B.M.
      • Bello S.H.
      • Sun Z.
      • McDonough M.A.
      • Oppermann U.
      • Schofield C.J.
      Inhibitor scaffolds for 2-oxoglutarate-dependent histone lysine demethylases.
      ,
      • Thalhammer A.
      • Mecinović J.
      • Loenarz C.
      • Tumber A.
      • Rose N.R.
      • Heightman T.D.
      • Schofield C.J.
      Inhibition of the histone demethylase JMJD2E by 3-substituted pyridine 2,4-dicarboxylates.
      ) and was recently shown to inhibit the JARID1B catalytic domain (
      • Kristensen L.H.
      • Nielsen A.L.
      • Helgstrand C.
      • Lees M.
      • Cloos P.
      • Kastrup J.S.
      • Helin K.
      • Olsen L.
      • Gajhede M.
      Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.
      ). Here we showed that 2,4-PDCA can also efficiently inhibit the JARID1 proteins, suggesting that it is a nonspecific demethylase inhibitor.
      In addition to the known inhibitors, we also identified several novel inhibitors. One such hit, named PBIT, inhibited JARID1B with a low micromolar IC50 value. PBIT is unlikely to be an iron chelator as similar IC50 values were obtained from experiments performed at both 15 μm and 50 μm Fe(II). True iron chelators are more effective at lower iron concentrations by scavenging much of the available iron. PBIT potently inhibits JARID1A/B/C, suggesting that it can act as a pan-JARID1 inhibitor. 10 μm PBIT has a minimal effect on the H3K27 demethylases UTX and JMJD3 (Fig. 4, D and E). In addition, the IC50 value of PBIT for JMJD2E is 28 μm (
      • King O.N.
      • Li X.S.
      • Sakurai M.
      • Kawamura A.
      • Rose N.R.
      • Ng S.S.
      • Quinn A.M.
      • Rai G.
      • Mott B.T.
      • Beswick P.
      • Klose R.J.
      • Oppermann U.
      • Jadhav A.
      • Heightman T.D.
      • Maloney D.J.
      • Schofield C.J.
      • Simeonov A.
      Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.
      ). Although we cannot exclude the possibility that PBIT also inhibits other JmjC demethylases and hydroxylases, our results suggest that PBIT is specific for the JARID1 enzymes.
      PBIT is a derivative of benzisothiazolinone, a widely used microbicide and fungicide used in many home cleaning products (
      • Dou D.
      • Alex D.
      • Du B.
      • Tiew K.C.
      • Aravapalli S.
      • Mandadapu S.R.
      • Calderone R.
      • Groutas W.C.
      Antifungal activity of a series of 1,2-benzisothiazol-3(2H)-one derivatives.
      ). PBIT and its analogues were previously identified as inhibitors of salicylate synthase from Mycobacterium tuberculosis (
      • Vasan M.
      • Neres J.
      • Williams J.
      • Wilson D.J.
      • Teitelbaum A.M.
      • Remmel R.P.
      • Aldrich C.C.
      Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening.
      ). The derivatives of benzisothiazolinone were explored as potential antiviral drugs by inhibiting enzymes such as macrophage migration inhibitory factor (
      • Jorgensen W.L.
      • Trofimov A.
      • Du X.
      • Hare A.A.
      • Leng L.
      • Bucala R.
      Benzisothiazolones as modulators of macrophage migration inhibitory factor.
      ). Therefore, many PBIT analogues are available and may inhibit the JARID1 enzymes. In fact, the PBIT analogue ebselen exhibited an IC50 of ∼6 μm against JARID1B (Table 1). Further studies optimizing the PBIT lead compound are currently ongoing. No crystal structure of the catalytic domains of the JARID1 enzymes has been published. It was shown recently that structure-guided virtual screen was essential to identify potent UTX inhibitors (
      • Kruidenier L.
      • Chung C.W.
      • Cheng Z.
      • Liddle J.
      • Che K.
      • Joberty G.
      • Bantscheff M.
      • Bountra C.
      • Bridges A.
      • Diallo H.
      • Eberhard D.
      • Hutchinson S.
      • Jones E.
      • Katso R.
      • Leveridge M.
      • Mander P.K.
      • Mosley J.
      • Ramirez-Molina C.
      • Rowland P.
      • Schofield C.J.
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      • Smith J.E.
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      • Thomas P.
      • Tumber A.
      • Drewes G.
      • Oppermann U.
      • Patel D.J.
      • Lee K.
      • Wilson D.M.
      A selective Jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response.
      ). Structural studies of the JARID1B enzyme with its inhibitors will be necessary to decipher their inhibitory mechanisms and to derive more potent inhibitors.
      PBIT treatment prevented the JARID1B overexpression-induced decrease of H3K4me3 in HeLa cells (Fig. 5). In addition, treatment of MCF7 cells with PBIT increased global levels of H3K4me3 (supplemental Fig. 3), suggesting that this compound is capable of entering the nucleus and inhibiting JARID1 H3K4 demethylases. Our cell-based assays showed that PBIT inhibited cell growth in a JARID1B level-dependent manner (Fig. 6, A–D). Consistent with these experiments, we showed that JARID1B knockdown decreased the proliferation of UACC-812 cells, but not MCF7 or MCF10A cells (Fig. 6, E–H). The effect of JARID1B knockdown on UACC-812 cells is not as dramatic as PBIT treatment, suggesting that either incomplete knockdown of JARID1B or functional compensation of JARID1A contributes to proliferation and survival of HER2-positive (HER2+) UACC-812 cells. JARID1B is overexpressed in HER2+ cells and human tumors,
      J. Cao and Q. Yan, unpublished data.
      suggesting that PBIT could be used to treat the HER2+ subtype of breast cancer. JARID1B knockdown was reported to decrease growth of MCF7 cells (
      • Yamane K.
      • Tateishi K.
      • Klose R.J.
      • Fang J.
      • Fabrizio L.A.
      • Erdjument-Bromage H.
      • Taylor-Papadimitriou J.
      • Tempst P.
      • Zhang Y.
      PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation.
      ). In contrast, similar to our studies, JARID1B knockdown did not affect the proliferation of MCF7 cells (
      • Scibetta A.G.
      • Santangelo S.
      • Coleman J.
      • Hall D.
      • Chaplin T.
      • Copier J.
      • Catchpole S.
      • Burchell J.
      • Taylor-Papadimitriou J.
      Functional analysis of the transcription repressor PLU-1/JARID1B.
      ). This discrepancy is likely due to different culture media used in these studies. Interestingly, PBIT treatment increased H3K4me3 level in MCF7 cells, but did not inhibit growth of these cells, suggesting that additional non-histone substrates of the JARID1 enzymes play critical roles in cell growth.
      JARID1A and JARID1B knock-out mice are viable (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ,
      • Klose R.J.
      • Yan Q.
      • Tothova Z.
      • Yamane K.
      • Erdjument-Bromage H.
      • Tempst P.
      • Gilliland D.G.
      • Zhang Y.
      • Kaelin Jr., W.G.
      The retinoblastoma binding protein RBP2 is an H3K4 demethylase.
      ,
      • Schmitz S.U.
      • Albert M.
      • Malatesta M.
      • Morey L.
      • Johansen J.V.
      • Bak M.
      • Tommerup N.
      • Abarrategui I.
      • Helin K.
      Jarid1b targets genes regulating development and is involved in neural differentiation.
      ), suggesting that inhibition of JARID1A or JARID1B has minimal effects on normal cells in vivo. JARID1A loss inhibits tumorigenesis in two mouse endocrine cancer models (
      • Lin W.
      • Cao J.
      • Liu J.
      • Beshiri M.L.
      • Fujiwara Y.
      • Francis J.
      • Cherniack A.D.
      • Geisen C.
      • Blair L.P.
      • Zou M.R.
      • Shen X.
      • Kawamori D.
      • Liu Z.
      • Grisanzio C.
      • Watanabe H.
      • Minamishima Y.A.
      • Zhang Q.
      • Kulkarni R.N.
      • Signoretti S.
      • Rodig S.J.
      • Bronson R.T.
      • Orkin S.H.
      • Tuck D.P.
      • Benevolenskaya E.V.
      • Meyerson M.
      • Kaelin Jr., W.G.
      • Yan Q.
      Loss of the retinoblastoma binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rb1 or Men1.
      ), suggesting that a JARID1A inhibitor could be used to treat these cancers. In addition, the tumors formed in the JARID1A knock-out mice showed increased JARID1B expression, implying that inhibitors that block both JARID1A and JARID1B enzymes are more effective in preventing tumor formation (
      • Lin W.
      • Cao J.
      • Liu J.
      • Beshiri M.L.
      • Fujiwara Y.
      • Francis J.
      • Cherniack A.D.
      • Geisen C.
      • Blair L.P.
      • Zou M.R.
      • Shen X.
      • Kawamori D.
      • Liu Z.
      • Grisanzio C.
      • Watanabe H.
      • Minamishima Y.A.
      • Zhang Q.
      • Kulkarni R.N.
      • Signoretti S.
      • Rodig S.J.
      • Bronson R.T.
      • Orkin S.H.
      • Tuck D.P.
      • Benevolenskaya E.V.
      • Meyerson M.
      • Kaelin Jr., W.G.
      • Yan Q.
      Loss of the retinoblastoma binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rb1 or Men1.
      ). The importance of JARID1 inhibitors will need to be confirmed in mouse models in which the endogenous JARID1 genes were replaced with the genes encoding catalytic inactive enzymes. Although we showed that the apparent JARID1 inhibitor PBIT has selective inhibitory activity on a HER2+ breast cancer cell line, the efficacy of PBIT and its derivatives on breast cancer needs to be further investigated with additional cell lines and in xenograft or genetically engineered mouse cancer models. As the JARID1 enzymes contribute strongly to tumorigenesis and drug resistance in multiple cancer types (
      • Blair L.P.
      • Cao J.
      • Zou M.R.
      • Sayegh J.
      • Yan Q.
      Epigenetic regulation by lysine demethylase 5 (KDM5) enzymes in cancer.
      ,
      • Hou J.
      • Wu J.
      • Dombkowski A.
      • Zhang K.
      • Holowatyj A.
      • Boerner J.L.
      • Yang Z.Q.
      Genomic amplification and a role in drug-resistance for the KDM5A histone demethylase in breast cancer.
      ),
      M. R. Zou, J. Cao, and Q. Yan, unpublished data.
      these inhibitors may also be effective for cancer therapy in those settings.

      Acknowledgments

      We thank Dr. Yi Zhang for providing FLAG-JARID1A and FLAG-JARID1B baculoviral constructs, Dr. Yang Shi for providing FLAG-JARID1C baculoviral construct, Drs. Kristian Helin and Stuart Orkin for providing His-FLAG-UTX baculoviral construct, and Dr. William Hahn for providing PLKO.1-shGFP construct. We thank Drs. Jon Morrow and Tian Xu for sharing their equipment. We thank members of the Bosenberg, Nguyen, Stern, and Yan laboratories for their kind help and valuable discussions. We thank Laura Abriola for technical help on hit validation and Amber Anders for help on generating the pcDNA3.1(−)-3×HA-JARID1B plasmid.

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